The long-range objective of this research project is the characterization of the calcium-binding proteins of nervous tissue in terms of their structure and function. We have isolated and purified to homogeneity two soluble calcium binding proteins from mammalian brain, CBP I and CDR. CDR is an activator of brain calcium-dependent cyclic nucleotide phosphodiesterase (PDE) and adenylate cyclase. The highest levels of this protein are found in electroplax of the electric eel, but CDR does not appear to activate PDE in this tissue, suggesting multiple roles for this protein. We will investigate the calcium and CDR dependence of several enzymatic processes in electroplax; these include adenylate and guanylate cyclase, cyclic nucleotide phosphodiesterase and protein kinase. It is not certain if CDR is a phosphoprotein; studies of protein phosphorylation in electroplax are designed to determine the endogenous substrates for protein kinases in this tissue as well as the cyclic nucleotide dependence of protein kinase activity in electroplax. In these studies, proteins of electroplax will be labeled in vitro with 32P and phosphate incorporation into individual peptides will be assessed by SDS-gel electrophoresis and autoradiography. We will attempt to purify and characterize the electroplax CDR binding protein which we have previously isolated, using affinity chromatography on columns of CDR-sepharose. Antibodies will be prepared to CBP I, CDR and CDR binding protein; this will make it possible to investigate the synthesis and phosphorylation of these proteins and make it feasible to develop radioimmune assays for their analysis. Membrane bound calcium-binding proteins of electroplax will be extracted with detergents and purified using procedures which have proven successful for the isolation of calcium-binding proteins from muscle membranes. Lastly, we will again address ourselves to CBP I, and seek to complete its chemical characterization and determine if it, like CDR, has enzyme activating properties.